CN102939161A

CN102939161A - A non contact real time micro polymerase chain reaction system and method thereof

Info

Publication number: CN102939161A
Application number: CN2011800219668A
Authority: CN
Inventors: 斯维萨·钱德拉; 苏迪普·蒙达尔; 文卡塔拉曼·文卡塔克里希南; 萨迪亚迪普·维斯瓦纳坦; 雷吉斯·马里拉德维·拉达克里希南; 拉维普拉卡什·贾亚拉曼; 钱德拉塞卡尔·巴斯卡兰奈尔; 皮拉里赛蒂·文卡塔·苏巴拉奥
Original assignee: Bigtec Pvt Ltd
Current assignee: Bigtec Pvt Ltd
Priority date: 2010-04-30
Filing date: 2011-04-28
Publication date: 2013-02-20
Anticipated expiration: 2031-04-28
Also published as: KR20130092391A; AR081288A1; TW201144432A; EP2563513A4; BR112012027876A2; WO2011135535A1; EP2563513A1; JP6026996B2; TWI591174B; JP2013524832A; US20130101983A1; EA201291008A1; SG185045A1; US9404151B2; CN102939161B

Abstract

The present disclosure provides a non contact real time micro Polymerase Chain Reaction [PCR] system comprises; a chip having a reaction chamber for holding a sample and an embedded metal heater below the reaction chamber for heating the sample; an optical unit comprising associated LED driver and photo detector amplifier placed above the chip to detect the fluorescence; an induction heater mounted around the chip and is inductively coupled to the metal heater; an infrared temperature sensor mounted below the chip for measuring a temperature of the metal heater, wherein said infrared temperature sensor is interfaced with a signal conditioner; and a controller interfaced with the signal conditioner and the induction heater for regulating the power to the induction heater based on feedback received from the infrared temperature sensor through the signal conditioner.

Description

A kind of contactless real-time little PCR system and method thereof

Technical field

Embodiments of the present invention relate to a kind of contactless real-time little PCR (PCR) system, more specifically, embodiments of the present invention relate to a kind of PCR system, and it has chip and infrared temperature sensor with the induction type heating of enclosed reaction chamber.

Background technology

In molecular biology, PCR be a kind of DNA (DNA) that makes single or several copies thus fragment amplification to some orders of magnitude produce 1,000,000 technology that copy of specific dna sequences.The method is based on thermal cycle, i.e. heating and cooling sample continuously.Along with the carrying out of PCR, the DNA that produces self is as copying template.PCR comprises three key steps of heating and cooling, repeats 25-30 time.Each circulation comprises: (i) in～94 degree sex change, this moment, all two strands was unwind into single stranded DNA; (ii) under the lower temperature of～54 degree, anneal, two primers and single-stranded template pairing this moment, (iii) extend at～72 degree, wherein polymerase extends primer by the base of connection and template complementation, forms thus the double-stranded DNA of two copies for each template.

Traditional Desktop PC R system uses large METAL HEATING PROCESS and cooling block to make the inner sample temperature circulation of loading of PA tube, and the miniaturization of reative cell has advantage aspect integrated, speed and the efficient.Because these advantages, the exploitation of the miniaturized system of PCR has been become the field of very paying close attention to.Many R﹠D teams have used the various materials that are suitable for biological respinse to produce device.Most R﹠D team has used silicon as its base material, because it has high heat conductivity and the processing conditions of superperformance.Naked silicon is that optics is opaque and report that it suppresses the PCR reaction.The silicon of oxidation or glass are preferred, but the processing of the multistep of the complexity of the reative cell in these chips, heater and temperature sensor makes it too expensive for the disposable application of special purpose.In addition, reative cell sealing step requires to avoid thermal evaporation, and this is usually by a kind of the finishing in the dissimilar Method for bonding, for example melt bonded method, anode Method for bonding, adhesive Method for bonding, reversible adhesive method and ultrasonic bonds method.On the other hand, polymer is the lower cost materials of biocompatibility, and it is transparent and easily at a lower temperature moulding.But its heat conductivity is compared much lower with silicon.Therefore the design of heater and temperature sensor is extremely important.

The selection of heating means and the selection of material are played important effect for quick microchip PCR scheme.In general, the normally used heating means of different team have two classes: contact and contactless heating.The contact heating method is utilized resistive heater heats PCR solution.Heater is film metal normally, is platinum (Pt) mostly, and this is because it has reproducible temperature dependency for resistance, bears the ability of high temperature, good chemical stability and high purity.In addition, usually use the thin layer of titanium as the adhesive layer of Pt, this is that this is so that its mis-behave because the latter at high temperature represents high diffusivity.But Pt is very expensive and optics is opaque.Other metal or alloy also has been used as heater.Some commercially available Po Er paste block thermoelectric units also have been widely used in the temperature control of pcr chip, although it has larger thermal mass, slower temperature slope and is opaque.The contactless heat protocol of the most frequently used PCR is based on hot air circulate, and it is finished by the temperature required air stream of quick switching.But for one single chip control and application hot-air not a duck soup.In some reports, use the infra-red radiation of tungsten lamp to be used as contactless thermal source, they need to be less than 15 minutes for 35 circulations.But tungsten lamp is noncoherent non-focusing light source, therefore needs high power (50-100W) so that chip reaches temperature required.In another report, the halogen lamp that uses non-costliness is used for the temperature of the little reative cell of silicon as the low power radiation source fast rise has been described.

According to discussion before, be necessary to research and develop so contactless real-time little PCR (PCR) system to overcome above-mentioned problem, this system have with the polymer chip of the induction type of enclosed reaction chamber heating and and infrared temperature sensor, this chip is made by the material that is selected from the group that includes but not limited to dimethyl silicone polymer (PDMS), acrylic acid series thing, polypropylene and Merlon.

Summary of the invention

Goal of the invention

An object of the present invention is to provide a kind of contactless real-time little PCR (PCR) system with induction heater and infrared radiation temperature sensor.

An object of the present invention is to provide a kind of contactless real-time little PCR (PCR) system that has with the chip of reative cell and embedded metal heater.

Summary of the invention

By providing such as system and method required for protection among the present invention, can overcome the shortcoming of prior art and extra advantage is provided.

Can realize characteristics and the advantage of adding by technology of the present invention.Herein other specific embodiment of the present invention and aspect are had been described in detail and be considered to the present invention's part required for protection.

The basic specific embodiment of the present invention provides a kind of contactless real-time little PCR (PCR) system.This system comprises the chip that has be used to the reative cell that holds sample and the metal heater that is used for heated sample that embeds below described reative cell.Comprise related led driver and the optical unit of photodetection amplifier, it places the chip top for detection of fluorescence.Metal heater is installed and be inductively coupled to induction heater around chip.Infrared temperature sensor is installed in the temperature that the chip below is used for measuring metal heater, and wherein said infrared temperature sensor engages with signal conditioner.And controller engages with signal conditioner and induction heater, to regulate the power of induction heater based on the feedback that receives from infrared temperature sensor by signal conditioner.

In a specific embodiment of the present invention, described chip by being selected from but the material that is not limited in the dimethyl silicone polymer (PDMS), acrylic acid series thing, Merlon, polyacrylic group make.

In a specific embodiment of the present invention, led driver and photodetector amplifier and pulse width module controller engage to monitor the different parameters of PCR with data acquistion and control system.

In a specific embodiment of the present invention, described induction heater is coil, and described coil is used for the eddy-current heating metal heater near described chip setting.And the distance between chip and coil changes between 0-10mm.

In a specific embodiment of the present invention, controller (5) is selected from least a in pulse width regulator, PID (PID) controller and the ON/OFF converter.

In a specific embodiment of the present invention, has nozzle in the sample filling process, to limit the air that is captured in a side of reative cell.

Another specific embodiment of the present invention provides the method for operating of a kind of contactless real-time little PCR (PCR) system.The method comprises sample is filled into behavior in the reative cell of chip.Use embedded metal heater heated sample, wherein said embedded metal heater is inductively coupled to induction heater to receive the heat energy from induction heater.Then use infrared temperature sensor to measure the temperature of metal heater.And the power of regulating induction heater with controller based on the feedback that receives from infrared temperature sensor.

In a specific embodiment of the present invention, the nozzle of the end by being arranged on described chip before filling sample washes away the air that captures from reative cell.

In a specific embodiment of the present invention, with comprising that related led driver and the optical unit of photodetection amplifier detect fluorescence.

In a specific embodiment of the present invention, Usage data collection and control system are monitored the different parameters of PCR.

In a specific embodiment of the present invention, supply with heat by induction heater to metal heater by the electromagnetic induction method.

Above for illustrative purposes only usefulness of general introduction is not to be intended in addition any restriction.Except above-described illustrative aspect, the specific embodiment and characteristics, further aspect, the specific embodiment and characteristics will be obvious by accompanying drawing and following detailed description.

Description of drawings

The characteristics of novelty of the present invention and characteristic are as claims are set forth.But the present invention self, preferred occupation mode, its further purpose and advantage also can get the best understanding by reference to the accompanying drawings by the detailed description with reference to the following specific embodiment.By embodiment the one or more specific embodiment is described referring now to accompanying drawing (the wherein identical identical element of Reference numeral representative), wherein:

Fig. 1 represents the decline block diagram of PCR system of noncontact.

Fig. 2 a-2c represents three step preparation processes with the PDMS chip of embedded heater and reative cell.

Fig. 2 d and 2e represent top view and the perspective view of PDMS chip.

Fig. 3 a-3c represents that coil is with respect to the position of chip, object lens and IR sensor.

It is that the diameter of 0.4mm is the 2D digital simulation of the magnetic field line of 8mm nickel ring that Fig. 4 represents around thickness, the copper coil eddy-current heating of the diameter 10mm that this nickel ring is made by the 1mm diameter wire with 2 circles, and load current 10A, frequency is 50kHz.

Fig. 5 represents the digital simulation of the function of the dissipated power of different heating material and frequency: stainless steel (SS), aluminium (Al) and copper (Cu).The dissipated power that has also shown copper coil self (coil).

Fig. 6 represents melting curve, and it shows the derivative of real-time fluorescence signal and the function of temperature (the calibration thermocouple of not calibrating the IR sensor reading and being used for the pipe of MJR thermal cycler inside of chip).

Fig. 7 represents the Temperature Distribution in the cyclic process.The black line represents by the instant set point that switches of computer, and red curve represents (corrected) temperature from the IR sensor.Left figure shows the completely distribution of whole circulations, and right figure represents former circulations.

The thermal cycler that Fig. 8 is illustrated in the PDMS chip real-time fluorescence signal of λ DNA of amplification 300bp and commodity in use carries out the real-time fluorescence signal of the λ DNA of amplification 300bp in the similar test in PA tube.

Fig. 9 is illustrated in the pipe and the curve analysis of the DNA of the 300bp that increases in the PDMS chip.Two sample pipetting volumes are passed through IR sensor record melting curve in chip.

The specific embodiment of the present invention usefulness for illustrative purposes only that accompanying drawing is described.Those skilled in the art will readily understand that the following explanation to described variation to structure and method of this paper can not deviate from described principle scope of the present invention herein.

The specific embodiment

Above characteristics of the present invention and technological merit have been carried out describing so that following detailed description of the invention is easier to understand roughly.Of the present invention its, his characteristics and advantage will be described and consist of the target of prescription of the present invention hereinafter.Those skilled in the art is to be understood that the disclosed specific specific embodiment can easily be utilized as the basis of improving or design other structure to finish identical purpose of the present invention with concept.Those skilled in the art should also realize that such equivalent structure does not deviate from essence and the scope of the present invention in claims.Those are considered to the characteristics of novelty of the present invention, no matter are about its composition or the aspect of method of operating, with further purpose and advantage, can be better understood according to following description taken in conjunction accompanying drawing content.But, only should understand clearly each accompanying drawing and be as an illustration the usefulness with description, and be not to be intended to limit the invention.

In order to overcome problem discussed above, the invention provides a kind of contactless real-time little PCR (PCR) system (A).This system comprises induction heater (2), and it is simple and without any need for light radiation and additional lens and filter.Eddy-current heating causes having the making step of simpler chip (1) of the reliability of raising.Further, use infrared temperature sensor (3) to be used for monitoring and control chip temperature in the system.This has been avoided using as the thermoelectric occasionally thermometer of the contact of resistor, and it need to use complicated making step to be embedded in the chip.Because thermometer is not the part of chip (1) self, but the cost of chip and disposability are greatly improved.Further, thermometer is as the part of chip reading unit, and its calibration only needs once just can finish, and does not need each chip is carried out.Remove outside the heating, using contactless temperature sense is to realize that fully complete contactless pcr chip advantage of system is necessary.

The invention provides the design of the complete contactless PCR system (A) that uses PDMS chip (1).Realize contactless heating by the use electromagnetic induction, and used Infrared Thermocouple (4) to finish contactless TEMP induction.More specifically, the present invention relates more specifically to: with the design of the chip (1) of embedded heater (1b); The optimization of the selection of heater metal and geometry thereof; Be used for the use of the infrared temperature sensor (3) of monitor temperature; The optimization of heater table finishing coat is beneficial to best infrared emission and fluorescence; Use the DNA melting curve with the calibration infrared thermometer; The enclosed reaction chamber (1a) that PDMS chip (1) is inner can use micro syringe that sample is injected wherein, to have avoided mineral oil or other additive demand as the volatilization vaporization stopping agent; Be used for heater (1b) parts and reative cell (1a) parts and use plasma bonding with the two processing technology that combines at air; The induction coil of resonant excitation, its frequency are tuned to can be to embedded heater (1b) through-put power best; At last, be used for based on low powder pulsed width adjusting (PWM) controller (5) from the power of the feedback regulation induction heater (2) of infrared temperature sensor (3).

The prototype that comprises the contactless PCR system (A) of fluorescence detection unit has made up and has used the amplification of λ DNA and unwind and test.PCR device of the present invention is per 0.5 second measurement fluorescence in the PCR process.The present invention has showed the amplification of the λ DNA that includes but not limited to 600bp, 800bp and 1000bp size, and has confirmed to compare the product specificity with melting curve with commercial PCR machine.Need not anyly to electrically contact and to pave the way for these low-cost systems of rapid deployment in different applications by the polymer chip that in real time microchip PCR uses, uses droppable transparent organism compatibility.

Method and material

Fig. 1 is the specific embodiment that exemplifies, and it has represented the noncontact PCR system (A) that declines.This system comprises the PDMS chip (1) with the embedded metal heater (1b) that is inductively coupled to induction heater (2).Infrared radiation temperature sensor (3) is from the temperature of the bottom HEATER FOR MEASURING (1a) of chip (1).Temperature signal and set point compare and regulate by PWM switch control circuit (5) power of supply induction heater (2).Optical pick-up unit (6) with related led driver and photodetection amplifier (7) places the top of chip (1a) to detect fluorescence.Photodetection amplifier (7) and PWM switch control circuit (5) engage with data acquistion and control system (8).Described data acquistion and control system (8) has the plug-in of the parameter of monitoring PCR.The further details of single subsystem will provide hereinafter.

The making of PDMS chip

Fig. 2 a-2c is the specific embodiment that exemplifies, and it has represented the step that relates in the making of pcr chip (1).The first half of chip (1) is comprised of reative cell (1a), and it is to use to copy the mold pressing fabrication techniques.At first design and use SU-8 and photoetch method to make mother matrix.Negative photoresist [SU82150] was spin-coated under the 1000rpm on one two inches the silicon chip in 30 seconds.After soft baking, the UV of the pattern on the photomask by two minutes exposed on the silicon chip that is delivered to the SU8 coating.Pattern after the exposure carries out rear baking, the then sclerosis baking in 30 minutes of developing.In order to obtain the first half of chip (1), 10: 1 PDMS are poured in the mother matrix, toast and peel off.The volume of reative cell (1a) is that about 5 μ L[～3mm diameter and 600 μ m are high].Side at circular cell has nozzle (1c) to limit the air that is captured in the sample filling process.The bottom of chip (1) is comprised of the thin circular metal plate that is embedded among the PDMS.This is to make by simply metal dish being immersed in the PDMS thin layer on the glass slide of pouring hydrophobic into., peel off with the thin slice cutting and from glass slide after 2 hours at 90 degrees centigrade of lower baking hardenings.At last, the first half of PDMS and the latter half are exposed in the air plasma 2 minutes, depress contact and place adding a little, then in baking box 90 degrees centigrade of lower heating 30 minutes to obtain the pcr chip (1) with the irreversible combination of embedded reactive chamber (1a) and heater (1b).

For using chip (1), reative cell (1a) at first uses micro syringe to locate to pierce through at nozzle (1c).Then use micro syringe to fill this chamber (1a) from a relative side with solution.Most air is overflowed to nozzle (1a) discharge and from the steam vent that pierces through.The air bubble that is captured of any remnants all is limited in the nozzle (1c) and can disturb PCR or fluorescence signal.

Fig. 2 d and 2e have represented respectively top view and the perspective view of PDMS chip (1).Chip (1) comprises be used to the reative cell that holds sample (1a) and the embedded metal heater (1b) that is used for heated sample below reative cell (1a).Chip (1) comprises that further the air that the nozzle (1c) of the side that is arranged in reative cell (1a) is used for capturing washes away from reative cell (1a).

The design of embedded heater

For given primary coil, the power that is coupled to the second level depends on resistivity and the physical dimension (diameter and thickness) of frequency, metal.Larger size can improve the power of coupling, has but reduced passive cooling rate owing to having increased thermal mass.Higher frequency can cause the better efficiency of heating surface, but must weigh the switching loss in the power transistor.Steel, copper and aluminum metal film to different commercially available different-thickness are attempted, and simulate to determine best switching frequency.Polish the end face of thin slice to such an extent that the very smooth fluorescence signal that can improve collection and bottom surface painted black can obtain best heat by infrared radiation temperature sensor (3) and detect.

Load coil and IR detector

Infrared detector (3) must be placed on～and 2mm is with interior temperature with the emitting surface of accurately experiencing diameter～7mm.It just in time is positioned in the below of chip (1) thus.

Because sensor main body is metal, the position of induction heater (2) primary coil is very important.When using the coil of 10 circles such as Fig. 3 chip that a is shown in (1) below, infrared temperature sensor (3) main body also is heated and the interference temperature signal.On the other hand, if when shown in Fig. 3 b, coil being placed chip (1) top, be heated and cause excessive power dissipation with the object lens (6) of metal master.And, to become inconvenience and cause the loss of the efficiency of heating surface of the processing of chip (1).Therefore, the design of final optimization pass is shown in Fig. 3 c, and it just in time arranges and form away from 3 circle coils of optical unit (6) and IR sensor (3) around chip (1) by one.Load coil changes between 0-10mm near the distance between chip (1) setting and induction coil and the chip (1).

Fluorescence detection unit

Optimize the fluorescence detecting system that is used for intercalating dye Sybr Green and formed by the blue LED excitaton source of 470nm, use the dichronic mirror projection and use the 20X micro objective to be focused on the PDMS chip (1).The emission of 520nm is collected by these same lens, uses bandpass optical filter to filter, and assembles at silicon photo diode.The intensity of LED is modulated onto 190Hz and detects synchronously light stream with the locking in the amplifier.Draw the signal that obtains each second by the time and obtain fluorescent emission continuous in the PCR process.

Temperature correction

Traditional IR sensor must advance calibration for the emissivity of the embedded heater of blacking.In addition, the reative cell (1a) that is positioned at away from heater (1b)～0.5mm will be under the lower temperature.Therefore, at the temperature standard of the inner DNA self that uses of reative cell (1a) as calibration.Specifically by the melting temperature with the inner DNA of fluorescence identification chip (1), record the signal of this IR sensor (3) when occuring that unwinds, and with the PCR thermal cycler that commercially obtains in the actual melting temperature that obtains compare to finish.By the DNA that use has different melting temperatures, sensor reading can be calibrated to the temperature of actual chamber.For given system (A), this only needs to do once, because subsequent reactions is to be same position in that similarly chip (1) is inner with respect to sensor.

Signal is processed and power control electronic equipment

DC signal from infrared radiation temperature sensor (3) amplifies with the thermocouple amplifier IC that the cold connection from analogue means (Analog Devices) compensates with amplifier buffer and use by accurate operation.Signal is transfused in the built-in comparator of the microcontroller that also can produce the PWM waveform.Design temperature from PC also is input in the comparator by USB DAC/ADC system.The output of comparator is transported to power MOSFET by mosfet driver control and described output with the form of pwm signal.Then transistor switch is opened, and will be provided to from the power of 5 volts of DC in the resonant tank that the low-power consumption electric capacity by induction heater primary coil and 10 μ F forms.The USB-DAQ system is recorded to the IR temperature signal on the PC, as from the fluorescence signal of optical unit.

Sample preparation

The PCR sample preparation is to use 2X DyNAmo Sybr green Master mix, Taq polymerase, λ DNA, PCR water and primer.With the λ dna profiling of 1 microlitre, the P1[5-AGT GTCGAA TTC TGATCG TGG TGA TAT CCG-3 of 2.5 microlitres], the P2[5-AGT GTC AAG CCT CAG CTT CAGTTC TCT-3 of 2.5 microlitres], the PCR water of the Master mix of the Taq polymerase of 1 microlitre, 25 microlitres and the 18 microlitres mixture of making 50 microlitres produces the DNA of the amplification of 311bp.With different primers produce～600bp and～the λ DNA of the amplification of 1000bp.

The execution in step of contactless PCR

Contactless real-time PCR system (A) uses the disposable PDMS chip (1) with embedded metal heater (1b) of sealing to move the PCR reaction, needs to carry out following steps:

A. the rules according to the available desk-top thermal cycler of common commerce prepare the PCR sample.Sample contains template DNA, primer, dNTPs, Taq polymerase or equivalent, applicable salt and the buffer in the PCR water.Sample also contains Sybr Green or Taqman fluorescence probe.Can use commercial Master mixs (for example DyNAmo kit) or its equivalent.

B. use clean and sterilized syringe with the fine needle head to locate to pierce through the air that reative cell (1a) is captured with release at nozzle (1c).Because disturbance reponse thereby this step are not optional to air bubble in some cases.

C. the sample with about 5 to 10 microlitres is expelled to by piercing through top seal in the reative cell (1a) of PDMS chip (1).The air that is captured in this process is pushed to nozzle (1c) and locates the hole of piercing through by step 2 and overflow.Any remaining air that is captured forms minute bubbles and can not disturb PCR subsequently to react in indent.

D. will place with the PDMS chip (1) of PCR sample in the small jig of coil inside of induction heater (2).The position of anchor clamps is interference of adjusting in advance to obtain best heating and fluorescence and can not being subject to any reaction.

E. optical head is reduced to the position of collecting fluorescence.The position of head is the interference of adjusting in advance to obtain maximum fluorescence and can not being subject to any reaction.

F. in some designs, optical head be fix and sample clamp is movably to place and moving chip (1).In final position, several millimeters of optical lens distance P DMS surface.

G. infrared radiation temperature sensor (3) is positioned at the position of adjusting in advance the sample clamp below and the interference that can not be subject to any reaction.Sample clamp has a centre bore so that sensor is collected infra-red radiation by this hole.

H. PDMS chip (1) and optical head (6) arrange in place after, provide an order with the executive software controller to microcomputer.The parameter of reaction, namely initial holding temperature and time, be used for also Input Software of cycle-index under each temperature and time of sex change, annealing and extension step and final elongating temperature and time.Then software is to the hardware issue an order of the temperature and time of each step of control.In each cyclic process, fluorescence is recorded continuously or with the number of times that the user determines.

I. after reaction, sample is cooled to room temperature.PDMS chip (1) can be shifted out from anchor clamps now.

J. the data of fluorescence and temperature function in time can be recalled and analyzed by software.

The result

The magnetic density that centers on the small coil generation of foil can be measured by QuickField 2-D electromagnetical analogies device, as shown in Figure 4.

For simulation, coil is 5.88x10 by electrical conductivity ⁷The conductor loop that the 1mm diameter copper of S/m is led thread two diameter 10mm forms.Heater is that electrical conductivity is 1x10 ⁷The diameter 8mm of S/m and the nickel dish of thickness 0.4mm and frequency are 50Hz.Except demagnetizing field, the function of the power with frequency that dissipates in copper coil (elementary) and the heater disk (second level) also calculates for different heating equipment material (being stainless steel (SS), aluminium (Al) and copper (Cu)), as shown in Figure 5.

Observe, the power that dissipates in elementary (coil) relatively is independent of material and increases with frequency.On the other hand, dissipate in the heater prominent be the Al under low frequency and under higher frequency, be stainless steel.But the power that consumes in switching transistor increases under higher frequency, is chosen in thus the Al heater (have with the similar electrical conductivity of copper and still have better fluorescent reflection in the surface of its polishing) under optimum frequency～130KHz in the PCR experiment.Best heater geometry is shaped as the Al sheet of thickness 0.38mm, diameter 7.14mm, and in the bottom side blacking to obtain the highest infrared emission.

Temperature correction be to finish by the melting curve from being loaded into from three kinds in the PA tube in the thermal cycler of the commerce of MJ Research USA different DNA (being λ DNA 300bp, 600bp and 1000bp) in chip (1) relatively.Loading about 5 microlitres and temperature rises to sex change (90 degree) oblique line from annealing (54 degree).

Fig. 6 has represented the melting curve that obtains, and table 1 contrasts the reading of the melting temperature of the DNA of IR sensor (3) in PDMS chip (1) with the actual melting temperature of reading from the thermal cycler of commerce.

Table 1: the melting temperature from pipe (thermocouple of calibration) and chip (unregulated IR sensor) of different λ DNA samples.

λDNA	Melting curve in the pipe	Melting curve in the chip
			300	81	67
600	83.5	68.4
			1000	88.5	73

The reading of the linear matched between the calibration curve of two foundation and IR sensor (3) is corrected to show the true temperature of the sample that the reative cell (1a) of follow-up PCR experiment is inner.Be used for the PCR rules that chip loads 5 μ L comprise initial under 96 degree 60 seconds denaturing step, follow by sex change (96 degree 15 seconds), annealing (48 degree 15 seconds) and extend (72 degree 100 seconds) step and carry out 18 circulations, and final spend downward-extensions 300 seconds 72.Fig. 7 has represented the Temperature Distribution that switch heater control is kept under from the feedback of contactless IR sensor (3).Need to prove, used the fan of a little～5W for improvement of cooling effectiveness when between denaturing step and annealing steps, switching.

The real-time fluorescence signal that Fig. 8 shows is from the amplification of 300bp λ DNA in the PDMS chip and uses the similar test of carrying out in the PA tube of commercial thermal cycler.

Amplification can repeatedly obtain for whole DNA sample (300bp, 600bp and 1000bp).After amplification, in same chip, carry out as shown in Figure 9 curve analysis.Also shown the melting curve that is obtained by the similar DNA sample that loads in another PDMS chip, but it is to increase in the pipe that uses commercial thermal cycler.Two operations of unwinding in the PDMS device almost are living, owing to different periods has small difference.

Commercial Application

The present invention can be widely used in the Bio-MEMS field,, uses the DNA cloning of PCR (PCR) that is.DNA cloning is used for the various forms of pathogen detection, legal medical expert's investigation, biophylaxis, food and water source control, environmental monitoring, dna sequencing etc.

Equivalent

About roughly any plural number and/or singular references used herein, based on context those skilled in the art can and/or use and translate into odd number and/or translate into plural number from odd number from plural number rightly.The change of different singular/plural should clearly be intended to the purpose clarified at this.

Those skilled in the art is to be understood that, in general, term used herein, particularly in claims (for example, the part in claims) (for example is intended for the term of open to the outside world usually, term " comprises " should be interpreted as " including but not limited to ", term " has " should be interpreted as " having at least ", term " comprise " and should be interpreted as " including but not limited to ", etc.).Those skilled in the art it is also understood that if be intended to quote certain optional network specific digit in front claim, its objective is to be explicitly recited in the claim, if lack such enumerating, then do not have such purpose.For example, for helping to understand, following claims can comprise uses introductory phrase " at least one " and " one or more " to introduce enumerating of claim.But, using such phrase must not mean by indefinite article " a " or " an " contains concrete claim that such claim enumerates and is limited to and only contains such invention of enumerating any, even if same claim comprises that introductory phrase " one or more " or " at least one " and indefinite article are such as " a " or " an " (for example, " a " and/or " an " ordinary solution is interpreted as " at least one " or " one or more "); Equally also should hold the application of the employed definite article of claim of enumerating for introduction.In addition, even enumerated clearly the claim of the concrete numeral of quoting, those skilled in the art also be to be understood that such enumerating usually should be interpreted as be at least the numeral enumerated (for example, only enumerated " enumerating for two " and do not had other modifications, usually mean at least two and enumerate, or two or more enumerating).Further, use " at least one among A, B and the C; etc. " the situation of usual pattern under, such explanation (for example can be understood this practice for a person skilled in the art in general, the system that includes but not limited to that " has at least one the system among A, B and the C " only have A, only have B, only have C, have A and B, have A and C, have B and C and/or have A, B and C, etc.).Those use " at least one among A, B and the C; etc. " the situation of usual pattern under, such explanation (for example can be understood this practice for a person skilled in the art in general, the system that includes but not limited to that " has at least one the system among A, B and the C " only have A, only have B, only have C, have A and B, have A and C, have B and C and/or have A, B and C, etc.).Those skilled in the art also should further understand when having two or more selectable term in fact any adversative conjunction and/or the phrase, no matter it is to appear in specification, claim or the accompanying drawing, all is interpreted as considering to comprise any one or two terms in one of term, the term.For example, phrase " A or B " should be understood to comprise the possibility of " A " or " B " or " A and B ".

Although herein disclosed is different aspects and the specific embodiment, other aspect and the specific embodiment are obvious for a person skilled in the art.Different aspect disclosed herein and specific embodiment usefulness for illustrative purposes only are not intended to limit, in the present claim of real scope and parenchymalia.

Reference numeral

Reference numeral	Explanation
		A	The PCR system
1	The PDMS chip
		1a	Reative cell
1b	Metal heater
		1c	Nozzle
2	Induction heater
		3	Infrared temperature sensor
4	Signal conditioner
		5	The PWM controller
6	Optical unit
		7	Led driver and photodetection amplifier
8	Data acquistion and control system

Claims

1. a contactless real-time little PCR (PCR) system (A), it comprises:

Chip (1), it has be used to the reative cell that holds sample (1a) and the embedded metal heater (1b) that is used for heating described sample below described reative cell (1a);

Optical unit (6), it comprises related led driver and photodetection amplifier (7), and places described chip (1) top for detection of fluorescence;

Induction heater (2), it installs and is inductively coupled to metal heater (1b) around described chip (1);

Infrared temperature sensor (3), it is installed in the temperature that described chip (1) below is used for measuring metal heater (1a), and wherein, described infrared temperature sensor (3) engages with signal conditioner (4); And

Controller (5), it engages with signal conditioner (4) and induction heater (2), to regulate the power of induction heater (2) based on the feedback that receives from infrared temperature sensor (3) by signal conditioner (4).

2. system according to claim 1, wherein, described chip is by at least a material manufacturing that is selected from dimethyl silicone polymer (PDMS), acrylic acid series thing, propylene and the Merlon.

3. system according to claim 1, wherein, described led driver and photodetection amplifier (7) and pulse width module controller (5) engage the different parameters with monitoring PCR with data acquistion and control system (8).

4. system according to claim 1, wherein, described induction heater (2) is coil, described coil arranges near chip (1) and is used for eddy-current heating metal heater (1a).

5. system according to claim 1, wherein, described controller (5) is selected from least a in pulse width regulator, PID (PID) controller and the ON/OFF converter.

6. system according to claim 1 wherein, has the air that nozzle (1c) is captured with restriction in described sample filling process in a side of reative cell (1).

7. the method for operating of a contactless real-time little PCR (PCR) system, described method comprises:

Sample is filled in the reative cell (1a) of chip (1);

Use embedded metal heater (1b) to heat described sample, wherein, described embedded metal heater (1b) is inductively coupled to induction heater (2) and is used for reception from the heat energy of induction heater (2);

Use infrared temperature sensor (3) to measure the temperature of metal heater (1b); And

Use controller (5) to regulate the power of induction heater (2) based on the feedback that receives from infrared temperature sensor (3).

8. method according to claim 7 wherein, is filled before the described sample by the nozzle in an end setting of chip (1) air that is captured is washed away from reative cell (1a).

9. method according to claim 7 wherein, is used to comprise that related led driver and the optical unit (6) of photodetection amplifier (7) detect fluorescence.

10. method according to claim 7, wherein, Usage data collection and control system (8) are monitored the different parameters of PCR.

11. method according to claim 7 wherein, is supplied with heat by induction heater (2) to metal heater (1a) by the electromagnetic induction method.

12. being used for being selected from DNA cloning, DNA extraction, cell analysis and chemical analysis/synthetic at least a BIO-MEMS, the system as claimed in claim 1 (A) uses.